My laboratory investigates the properties of natural and chemically modified nucleic acids. Working at the interface of cell biology and nucleic acid chemistry we have modified cellular activities including transcription, translation, allele-selectie inhibition of protein expression, splicing, and telomerase-mediated elongation of telomeres. For 2016-2020 our central goal is to examine the recognition of cellular RNA with a primary focus on nuclear RNA. We will apply that understanding to methods for controlling gene expression and new insights into natural pathways of gene regulation. Much of the genome is transcribed into RNAs that do not encode proteins. The function of many noncoding RNAs is unclear, as is the mechanism for how they might affect gene expression. Published reports offer little insight into the molecular details for how a specific noncoding RNA affects expression of a given gene. In the absence of this information, validation of proposed effects becomes problematic and the predictive power for studying novel genes is limited. This lack of guiding mechanistic principles is a major obstacle to progress. RNAi provides a potential mechanism for recognizing specific nuclear RNA species. While RNAi is well-known as a driving force for recognition of mRNA in the cytoplasm of mammalian cells, its potential to drive recognition in the somatic cell nuclei has been unclear. We propose to define the scope and mechanism of mammalian nuclear RNAi and to apply it to novel disease targets. Objective 1. Understand nuclear RNAi. We will characterize the protein and RNA interactions involved in nuclear RNAi. RNAseq will be used to identify RNA targets for functional validation. Mass spectrometry will be used to determine protein partners. We will explore the mechanism of nuclear RNAi and obtain insights into the how nuclear RNAi regulates gene expression in mammalian cells. These data will reveal how argonaute proteins and other RNAi factors function in the nucleus to control gene expression. Objective 2. Novel targets for nucleic acids inside cells. We will also expand recognition to novel nucleic acids targets including the expanded intronic repeats within the mutant frataxin and C9orf72 genes. These data will further develop our understanding of the mechanism of nuclear RNAi and show how nuclear RNAi can be applied to the control of disease gene expression. Compounds that control frataxin protein expression or disrupt structures formed by the expanded repeat within intronic C9orf72 would be lead compounds for drug development.